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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
one-generation reproductive toxicity
Remarks:
based on generations indicated in Effect levels (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 3 July 2012 to 14 November 2012
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Well documented GLP study performed according to OECD Guideline 422. However, dose formulations were not analyzed.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
no analytical verification of the dose is performed
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): N-Methylmorpholine Oxide 50 (50% aqueous solution)
- Substance type: Organic
- Physical state: Liquid
- Lot/batch No.: F30-T151-291211
- Storage condition of test material: at room temperature protected from humidity and light

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: BIOAGRI Laboratorios
- Age at study initiation: 11 weeks old
- Weight at study initiation: range: males: 344.7 to 361.8 grams; females: 203.3 to 220.9 grams; satellite males: 330.0 - 338.0 grams; satellite females: 221.2 - 227.0 grams
- Fasting period before study: no
- Housing: Each animal was housed individually, except during cohabitation. After acclimatisation, one male was placed into each cage for pairing. After pairing, females that presented vaginal smears with the presence of sperm were considered mated and housed individually. Rats were housed in polypropylene cages (41 x 34 x 19 cm) with wire mesh tops and bedding material (wood shavings). Clean cages were provided twice weekly for all animals. The cages with the test animals were arranged on the racks in such a way that uniform experimental conditions (ventilation and light) were ensured.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.2 - 24.6°C
- Humidity (%): 37.7 - 70.0%
- Air changes (per hr): 10-20 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: deionized water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
For each dosage group, the appropriate amount of N-Methylmorpholine Oxide 50 was weighed into a pre-calibrated beaker. The vehicle (deionized water) was added in sufficient quantity to achieve the desired concentration. Each solution was stirred and dispensed into individual containers properly identified. A sufficient quantity of the vehicle was similarly dispensed for administration to control animals. The prepared solutions were stored at room temperature.

Test solutions were prepared daily at the testing facility and were administered within 2 hours after preparation. The test solutions were stirred automatically during administration to maintain the homogeneity.
Details on mating procedure:
- Premating: At the end of the acclimation period, animals were randomly assigned to the experimental groups, housed and the treatment started.
- Mating: After a premating period of 2 weeks, females were cohabited with an assigned male (1 female: 1 male) from the same dose level until evidence of copulation was observed. Care was taken to avoid sibling mating. Vaginal smears were collected daily durng the mating period and examined for the presence of sperm. Day 0 of gestation was defined as the day sperm is found in the vaginal smear. Males were euthanized after completing a dosing period of 36 to 37 days.
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
Female: from 14 days before mating period to 4 days post-delivery, animals were doses with a volume of 4mL/kg bw.
Male: after completing a dosing period of 36 to 37 days, animals were doses with a volume of 4mL/kg bw.
Frequency of treatment:
7-day-week-basis
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 10, 100 and 1000 mg/kg/day
Basis:
actual ingested
as 50% solution
No. of animals per sex per dose:
12 and 5 for the satellite group
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale: The dosage levels (in mg/kg body weight/day) were selected on the basis of previous studies with the test article.


Examinations

Parental animals: Observations and examinations:
DETAILED CLINICAL OBSERVATIONS: Yes
- All animals underwent daily clinical observation for overt signs of ill health. These include, but arenot limited to, changes in skin and fur, eye and mucous membranes, respiratory, circulatory, autonomic and central nervous system, motor activity and behavioral patterns.

BODY WEIGHT: Yes
- Males were weighed on the first day of dosing and weekly therafter including mating and post-mating periods). Females were weighed on first day of dosing and once a week during premating and mating periods, on days 0, 7, 14 and 20 of gestation, and during lactation on the same days as the weighing of litters (on days 0 and 4 postnatal).

FOOD CONSUMPTION:
Food consumption was determined on the same day of body weight determination during premating and lactation periods, except on day 0. During gestation period food consumption was determined on days 3, 6, 9, 12, 15, 18 and 20. After mating period, food consumption of males was determined weekly. Food consumption was not determined during the mating period.

HAEMATOLOGY: Yes
Hematology parameters:
red blood cell count, hemoglobin, hematocrit, platelets, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, total white blood cell count, differential leukocyte count (band neutrophils, monocytes, segmented neutrophils, lymphocytes, eosinophils, basophils)
clotting parameters: prothrombin time, activated partial thromboplastin time.

Hematology determinations were performed on 5 parental animals/sex/group, randomly selected from each group, and on satellite animals from control and high dose groups. The animals were fasted overnight and anesthetized by CO2 prior to blood collection (cardiac puncture).

CLINICAL CHEMISTRY: Yes
aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, total protein, albumin, glucose, total cholesterol, urea nitrogen, creatinine, sodium, potassium, calcium, globulin, albumin/globulin ratio.

Clinical chemistry determinations were performed on 5 parental animals/sex/group, randomly selected from each group, and on satellite animals from control and high dose groups. The animals were fasted overnight and anesthetized by CO2 prior to blood collection (cardiac puncture).

NEUROBEHAVIOURAL EXAMINATION: Yes
Functional observational battery:
Sensory reactivity to stimuli and motor activity assessment were performed on 5 animals/sex/group on males at the end of the dosing period, before scheduled necropsy, and on females during lactation. The following parameters were assessed:
A - Autonomic functions: lacrimation, salivation, palpebral closure, prominence of the eye, piloerection and respiration;
B - Reactivity and sensitivity: sensor motor responses to approach tactile and tail flick;
C - Excitability: reactions to handling and behavior in an open field;
D - Gait and sensor motor coordination: degree of mobility and gait pattern in an open field;
E - Abnormal clinical signs: including convulsions, tremors, unusual behavior and deposits around the eyes, nose or mouth

OTHER: ORGAN WEIGHTS
At scheduled necropsy, testes and epididymes of all males were weighed. Organs weights were obtained for the following organs from 5 animals/sex/group: liver, kidneys, adrenals, thymus, spleen, brain and heart.
Oestrous cyclicity (parental animals):
No data
Sperm parameters (parental animals):
testis weight, epididymides weight
Litter observations:
Live pups were counted, sexed and weighed on days 0 and 4 postnatal.
Postmortem examinations (parental animals):
GROSS PATHOLOGY: Yes
Females that did not deliver were euthanized 24-26 days after the last day of mating period.
At termination, all parental animals were examined macroscopically for any abnormalities or pathological changes. The animals were euthanized in a carbon dioxide chamber. The numbers of implantation sites and corpora lutea were recorded. The animals were disposed in biological garbage and then incinerated. All pups were grossly examined for abnormalities of the oral, thoracic and abdominal cavities.

HISTOPATHOLOGY: Yes
At scheduled necropsy, the following organs of all animals were preserved:
testes, epididymides, ovaries, prostate, seminal vesicle and coagulating gland, bulbourethral gland, organs showing alterations

The following organs and tissues of 5 animals/sex/group were preserved:
adrenals (right and left), bone marrow (femur), brain (cerebrum, cerebellum and pons), esophagus, heart, intestine (duodenum, jejunum, ileum - including Peyer's patches, colon, cecum, rectum/anus), kidneys (right and left), liver (3 lobes), lungs, lymph nodes (mesenteric and submaxillary), pancreas, peripheral nerve (sciatic), salivary gland (right and left), spinal cord (cervical, midthoracic and lumbar sections), spleen, stomach (glandular and non-glandular), trachea, thymus, thyroid/parathyroid, urinary bladder, uterus, all gross lesions

Full histopathology of the preserved organs and tissues listed above were performed in highest dose and control animals, and, kidneys and stomach from two female rats, No 52 and 73, exposed to 10 and 100 mg/kg/day, respectively.
Postmortem examinations (offspring):
SACRIFICE
All pups were euthanized at day 4 postnatal

GROSS NECROPSY
All pups were grossly examined for abnormalities of the oral, thoracic and abdominal cavities.
Statistics:
Quantitative variables such as body weights, food consumption and organ weights were analyzed by One Way Analysis of Variance (ANOVA), followed by Dunnett's test if significance is detected, or by non-parametric test of Kruskal-Wallis, according to the results of tests for normality and homogeneity of variance. For qualitative or non-parametric data such as clinical findings, macroscopic and microscopic findings and fetal findings, comparison between means were carried out using the Fisher's Exact Test or Chi-Square Test. The level of significance was set at 5%.
Reproductive indices:
The precentage of pre-implantation loss, post-implantation loss, mating index, fertility index, gestation index on day 4 post-partum were calculated (for each pregnant animal) according to the following:
% Pre-implantation loss = (Number of corpora lutea - Number of implantation sites x 100)/Number of corpora lutea
% Post-implantation loss = (Number of implantations - Number of live fetuses x 100)/Number of implantations
% Mating index = (Number of females mated x 100)/Number of females paired
% Fertility index = (Number of females pregnant x 100)/Number of mated pairs
% Gestation index = (Number of females with live pups at birth x 100)/Number of females pregnant
Offspring viability indices:
The percentage of live birth index and viability index on day 4 post-partum were calculated (for each pregnant animal) according to the following:
% Live birth index = (Number of live born pups x 100)/Number of delivered pups
% Viability index on day 4 post-partum = (Number of surviving pups on day 4 post-partum x 100)/Number of live born pups

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
effects observed, treatment-related

Details on results (P0)

CLINICAL SIGNS AND MORTALITY
No mortality and no clinical signs were observed for any group.

BODY WEIGHT, BODY WEIGHT GAIN AND FOOD CONSUMPTION
Treatment related effects at the high dose level (1000 mg/kg bw) were observed in body weight, body weight gain and food consumption of male and female rats, where a statistically significant decrease was noted during the entire treatment period. However, during the recovery period, treated satellite males and females showed an increase in all these indicators, which are considered to be reversible, since 14 days after treatment, treated satellites had higher body weight, body weight gain and food consumption than the control group.

HAEMATOLOGY
No treatment-related effects were observed in hematology and clotting parameters.

CLINICAL CHEMISTRY
No treatment-related effects were observed in clinical chemistry parameters.

NEUROBEHAVIOUR
No treatment-related findings were observed in the functional observation battery.

ORGAN WEIGHTS
No treatment-related effects were observed in organ weights.

GROSS PATHOLOGY AND HISTOPATHOLOGY: NON-NEOPLASTIC
No treatment-related findings were observed during necropsy or microscopic examination in male or female rats

REPRODUCTIVE FUNCTION AND PERFORMANCE:
The mean number of days of pairing before mating and the length of the gestation period were comparable between the control and treated groups and within the physiological range of this species and strain.
For the control group, all females were pregnant, while 3/12, 2/12 and 1/12 were not pregnant at 10, 100 and 1000 mg/kg/day groups, respectively. The mating index was 100% in all groups, and gestation indices were 91.6%, 75.0%, 75.0% and 91.6% at the dose-levels of 0, 10, 100 and 1000 mg/kg/day, respectively.
The live birth index at 100 and 1000 mg/kg/day was lower than control (-3.7% and -3.2%), although very small in magnitude. However, the viability index on day 4 post-partum was slightly lower at 1000 mg/kg/day (-12.8%) when compared to the control group. This finding at the high dose level was related to a lower implantation number (-23.8%) and statistically lower number of corpora lutea (- 28.9%) and total number of pups (-30.3%) and live pups (-32%). On post-partum day 4, at 1000 mg/kg/day a statistically significant lower total number of pups (-32%) and live pups (-41%) was noted compared to the control group. These effects were considered to be treatment-related and with toxicological significance.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: overall effects; reported dose is 100 mg/kg bw/d as 50% solution
Dose descriptor:
LOAEL
Effect level:
500 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: body weight; food consumption; reported dose is 1000 mg/kg bw/d as 50% solution

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Histopathological findings:
not examined

Details on results (F1)

VIABILITY (OFFSPRING)
On post-partum day 0, the number of pups found dead was statistically higher at 100 and 1000 mg/kg/day (+3.6% and +3.1%, respectively) compared to the control group. Despite statistical significance, these differences were very small in magnitude and not dose related. However, on post-partum day 4, at 1000 mg/kg/day the number of pups found dead was statistically significantly higher than control (+12.8%). Since this event occurred at the high dose level, it was considered to be test article relatied and evidence of slight toxicity of the test article, associated with maternal toxicity.

BODY WEIGHT (OFFSPRING)
Statistically significant lower body weight was observed in pups from dams exposed to 1000 mg/kg/day (-17.3%) when compared to the control group.

GROSS PATHOLOGY (OFFSPRING)
Cyanosis and body trauma (+8.8%) and cannibalized pups (+10.2%) were findings statistically higher at the highest dose level with a dose-related trend. These effects, associated with maternal toxicity, were observed at this highest dose and could be attributed to treatement with the test item.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: embryo-fetal toxicity; reported dose is 100 mg/kg bw/d as 50% solution.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Under the experimental conditions of this study, the NOAELs are 50 mg/kg/d for both parental fertility and reproduction and for offspring toxicity. It is assumed that the effects on the offspring are secondary to maternal toxicity. To verify this assumption, additional tests are proposed.